In view of the observed ET-1 stimulation of ion transport systems in RBEC, it has been important to establish whether these systems also exist in microvascular endothelium derived from human brain (HBMEC). Uptake of 86Rb+ (0.2 gammaC/well) as a tracer of K+ uptake was determined in confluent HBMEC preincubated (30 min) with inhibitor or antagonist and incubated (5 min) alone or with ET-1 or ET-3 in serum-free medium at room temperature. The same procedures were used for HBMEC exposed to hypoxia (95% N2, 5% CO2) for 24 hr. ET-1 but not ET-3 dose-dependently increased K+ uptake which was inhibited with BQ123 (ET/A receptor antagonist) but not with IRL1038 (ET/B receptor antagonist). Ouabain (Na+K+-ATPase inhibitor) reduced the ET-1-stimulated K+ uptake to a greater degree (94%) than bumetanide [Na+K+Cl- cotransport inhibitor) 30%. N-ethyl-n- isopropyl amelinide (EIPA) the inhibitor of N+/H+ exchange reduced the ET-1-stimulated K+ uptake in the presence of bumetanide only. Verapamil, the inhibitor of Ca2+ channels decreased the ET-1 stimulated K+ uptake in the presence of ouabain but not with bumetanide. In contrast, staurosporine [inhibitor of protein kinase c (PKC)] reduced the ET-1 stimulated K+ uptake in the presence of bumetanide but not ouabain. Overnight exposure of HMBEC in nitrogen atmosphere dose-dependently augmented the ET-1 stimulation of K+ uptake affecting the ouabain- sensitive K+ uptake only. The data indicated that: 1) Na+K+-AT Pase activity and Na+K+Cl- cotransport are stimulated by ET-1 through activation of ET/A receptors in HBMEC; 2) the ET-1 stimulation of the Na+K+-AT Pase activity is mediated by Ca2+ ions and is linked to Na+/H+ exchange, whereas the Na+K+Cl- cotransport is linked to PKC; and 3) hypoxia amplifies the ET stimulation of Na+K+-AT Pase activity. This study represents the first demonstration of ionic transport systems in HBMEC. The observed ET-1 modulation of Na+K+-ATPase activity and Na+K+Cl- cotransport indicate that ET-1 may play a role in regulating electrolytes transport across the blood-brain barrier (BBB). The hypoxic augmentation of ET-1 stimulated Na+K+-ATPase activity strongly suggests that ET-1 (released from vascular, blood and/or brain cells) may participate in the disturbances of water electrolytes homeostasis under pathological conditions such as ischemia.